In vitro expression of the restriction endonucleases LlaMI and ScrFI isolated from Lactococcus lactis M19 and UC503.

A new restriction endonuclease LlaMI has been characterized in Lactococcus lactis subsp. cremoris M19. LlaMI recognizes the sequence 5'-CCNGG-3' and cuts after the second cytosine. This restriction endonuclease is related to commercially available ScrFI but not identical to it. Comparative analysis of the predicted amino acid sequences of LlaMI and ScrFI indicates five non-conservative amino acid changes between these two restriction enzymes. These two enzymes were expressed in vitro as histidine-tagged fusion proteins. LlaMI was shown to be more sensitive to high salt concentration than ScrFI. Southern blotting and hybridization analysis indicate that the gene for LlaMI R/M system is chromosomally encoded.

A rapid method combining immunofluorescence and flow cytometry for improved understanding of competitive interactions between lactic acid bacteria (LAB) and methicillin-resistant S. aureus (MRSA) in mixed culture.

The increasing frequency of methicillin-resistant Staphylococcus aureus (MRSA) infections in hospital and community settings highlights the need for effective anti-MRSA agents that will not contribute to the growing problem of antibiotic resistance. Lactic acid bacteria (LAB) are known to exclude various pathogens through multiple mechanisms. In vitro models studying interactions of pathogens and LAB in mixed cultures use selective agar plates to quantify changes in target populations. We applied commercially available S. aureus-specific polyclonal antibodies conjugated with fluorescein isothiocyanate (FITC) for this purpose, producing a bright green signal that clearly differentiates S. aureus from LAB species when mixed cultures are analyzed by flow cytometry and fluorescent microscopy. Flow cytometry of mixed cultures revealed a much larger population of MRSA cells than was detectable using selective agar plates. To our knowledge, this is the first time immunofluorescent flow cytometry has been applied to the study of competitive exclusion in mixed bacterial populations over time.

Combined effect of gamma irradiation, ascorbic acid, and edible coating on the improvement of microbial and biochemical characteristics of ground beef.

This study was conducted to evaluate the combined effect of gamma irradiation and the incorporation of natural antimicrobial compounds in cross-linked films on the microbiological and biochemical characteristics of ground beef. Medium-fat (23% fat) ground beef patties were divided into three separate treatment groups: (i) control samples without additives, (ii) ground beef samples containing 0.5% (wt/wt) ascorbic acid, and (iii) ground beef samples containing 0.5% ascorbic acid and coated with a protein-based cross-linked film containing immobilized spice powders. Meat samples were irradiated at doses of 0, 1, 2, and 3 kGy and stored at 4 +/- 2 degrees C. Microbial growth (based on total aerobic plate counts [APCs] and total coliforms) was evaluated, as were the content of thiobarbituric acid-reactive substances (TBARS) and that of free sulfydryl groups. At the end of the storage period, Enterobacteriaceae, presumptive Staphylococcus aureus, presumptive Pseudomonas spp., Brochothrix thermosphacta, and lactic acid bacteria were enumerated. Regardless of the treatment group, irradiation significantly (P < or = 0.05) reduced the APCs. Irradiation doses of 1, 2, and 3 kGy produced immediate APC reductions of 2, 3, and 4 log units, respectively. An APC level of 6 log CFU/g was reached after 4, 7, and 10 days for samples irradiated at 1, 2, and 3 kGy, respectively. Lactic acid bacteria and B. thermosphacta were more resistant to irradiation than were Enterobacteriaceae and Pseudomonas. The TBARS and free sulfydryl contents were stabilized during postirradiation storage for samples containing ascorbic acid and coated with the protein-based cross-linked film containing immobilized spice powders.

Role of probiotics in the prevention and treatment of meticillin-resistant Staphylococcus aureus infections.

Meticillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant micro-organism and is the principal nosocomial pathogen worldwide. Following initial in vitro experiments demonstrating that Lactobacillus acidophilus CL1285(®) and Lactobacillus casei LBC80R(®) commercial strains exhibit antibacterial activity against clinical MRSA isolates, we conducted a literature search to find any evidence of probiotic efficacy in decolonisation or treatment of S. aureus infection. As summarised below, many strains of lactobacilli and bifidobacteria isolated from a variety of sources inhibited the growth of S. aureus and clinical isolates of MRSA in vitro. The most active strains were Lactobacillus reuteri, Lactobacillus rhamnosus GG, Propionibacterium freudenreichii, Propionibacterium acnes, Lactobacillus paracasei, L. acidophilus, L. casei, Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus fermentum and Lactococcus lactis. Their effects were mediated both by direct cell competitive exclusion as well as production of acids or bacteriocin-like inhibitors. L. acidophilus also inhibited S. aureus biofilm formation and lipase production. In vitro antimicrobial activity did not necessarily assure efficacy in vivo in animal infectious models, e.g. S. aureus 8325-4 was most sensitive in vitro to L. acidophilus, whilst in vivo Bifidobacterium bifidum best inhibited experimental intravaginal staphylococcosis in mice. On the other hand, L. plantarum, which showed the highest inhibition activity against S. aureus in vitro, was also very effective topically in preventing skin wound infection with S. aureus in mice. Very few clinical data were found on the interactions between probiotics and MRSA, but the few identified clinical cases pointed to the feasibility of elimination or reduction of MRSA colonisation with probiotic use.

Antibacterial activity of Lactobacillus acidophilus and Lactobacillus casei against methicillin-resistant Staphylococcus aureus (MRSA).

Methicillin-resistant Staphylococcus aureus (MRSA) is a multidrug-resistant microorganism and the principal nosocomial pathogen worldwide. The antibacterial activity of lactic acid bacteria against MRSA from ten human clinical isolates as well as MRSA standard strain ATCC 43300 was tested in vitro. The Lactobacillus (Lb.) strains (Lb. acidophilus CL1285(®) and Lb. casei LBC80R) as pure cultures, which came from commercial food products were employed. The growth inhibitory effect produced by the antimicrobial activity of the lactic acid bacteria on the MRSA strains was tested on solid medium using agar diffusion methods as well as a using a liquid medium procedure that contained a mixture of MRSA and lactic acid bacteria cultures. In the latter instance, we were able to demonstrate that the direct interaction of lactic acid bacteria and MRSA in such a mixture led to the elimination of 99% of the MRSA cells after 24 h of their incubation at 37°C.

The Effect of Various Probiotic Strains or Avilamycin Feed Additive on Immune Defense Markers and Acute-Phase Response to Salmonella Infection in Chickens.

Probiotics are a nutritional tool for disease prevention. It has been proposed that stimulation of immune response could affect the growth-promoting properties of antimicrobial growth promoters as well as the control of foodborne pathogens. The current study compares immune response in the blood of 280 non-infected and Salmonella-infected chickens fed either with the growth promoter avilamycin or with one of five probiotic strains of Lactobacillus and Bifidobacterium, which also showed growth-promoting properties. All of the probiotic strains stimulated superoxide anion production and the proliferation of leukocytes, while raising lysozyme and γ-globulin levels (by up to 65%, p < 0.01), which are important factors in native and cell-mediated immune defense against pathogens. In contrast, among the two strains examined, specific Salmonella antibodies were induced only by L. salivarius, and not by B. animalis, as assessed by the ELISA method and confirmed by an agglutination reaction (p < 0.05). In the avilamycin-fed group, both non-infected and infected chickens showed decreased levels of these immune markers (by 30%) and increased levels of ceruloplasmin by up to 35%. In contrast, the probiotics suppressed acute-phase response assessed by ceruloplasmin by up to 32%. This correlation implies that various antimicrobial feed additives have a distinct effect on immunomodulation, which may affect different mechanisms in the nutrition-related metabolism associated with the rate of weight gain in chickens. The data could contribute to the design of innovative antimicrobial feed additives in the food industry and consequently to well-being of humans.